Involvement of elevated proline accumulation in enhanced osmotic stress tolerance in Arabidopsis conferred by chimeric repressor gene silencing technology

Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATH B23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PDH) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PDH gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.